Composite Reference Standard Research Articles

Sensitive Diagnosis and Post-Treatment Follow-Up of Schistosoma mansoni Infections in Asymptomatic Eritrean Refugees by Circulating Anodic Antigen Detection and Polymerase Chain Reaction.

ABSTRACT.The increasing number of refugees coming from or passing through Schistosoma-endemic areas and arriving in Europe highlights the importance of screening for schistosomiasis on arrival, and focuses attention on the choice of diagnostic test. We evaluate the diagnostic performance of circulating anodic antigen (CAA) detection in 92 asymptomatic refugees from Eritrea. Results were compared with already-available stool microscopy, serology, and urine point-of-care circulating cathodic antigen (POC-CCA) data. For a full diagnostic comparison, real-time polymerase chain reaction (PCR) and the POC-CCA were included. All outcomes were compared against a composite reference standard. Urine and serum samples were subjected to the ultra-sensitive and highly specific up-converting particle lateral flow CAA test, Schistosoma spp. real-time PCR was performed on urine and stool, and the POC-CCA was used on urine using the G-score method. CAA was detected in 43% of urine and in 40% of serum samples. Urine PCR was negative in all 92 individuals, whereas 25% showed Schistosoma DNA in stool. POC-CCA was positive in 30% of individuals. The CAA test confirmed all microscopy positives, except for two cases that were also negative by all other diagnostic procedures. Post-treatment, a significant reduction in the number of positives and infection intensity was observed, in particular regarding CAA levels. Our findings confirm that microscopy, serology, and POC-CCA lack the sensitivity to detect all active Schistosoma infections. Accuracy of stool PCR was similar to microscopy, indicating that this method also lacks sensitivity. The CAA test appeared to be the most accurate method for screening active Schistosoma infections and for monitoring treatment efficacy.

Diagnostic utility of chest computerized tomography in the diagnosis of recurrence among sputum scarce and sputum negative previously treated pulmonary tuberculosis suspects.

Objective:The objective was to study the sensitivity, specificity, and diagnostic accuracy of various computed tomography (CT) chest findings in diagnosing recurrence among pulmonary tuberculosis (PTB) suspects.Materials and Methods:A prospective observational study was conducted in a tertiary care hospital in New Delhi. A total of 130 suspects with a past history of treatment for PTB, who presented with any of the symptoms suggestive of recurrence were included. Sputum-positive, HIV-positive patients, pregnant females, and patients aged <18 years were excluded. Patients underwent CT chest followed by bronchoalveolar lavage (BAL).Results:A total of 62 patients were there in the final analysis. The median age of the patients with recurrent PTB was 27.5 years. Cough was the universal symptom in all these patients (>90%). Hemoptysis was the predominant symptom among patients with chronic pulmonary aspergillosis (66.6%). Necrotic mediastinal lymph nodes had good diagnostic accuracy of 88.71% with area under the curve of 0.806, P < 0.001 in diagnosing recurrent TB. BAL GeneXpert and mycobacteria growth indicator tube had good sensitivity (83.33% and 84.62%, respectively), specificity (100% for both), and excellent diagnostic accuracy (95.16% and 96.36%, respectively) for diagnosing recurrence in sputum negative and sputum scarce patient, (P < 0.001) when compared with composite reference standard. For culture-positive cases, BAL GeneXpert MTB/RIF had 100% sensitivity and 97.73% specificity in diagnosing recurrent PTB patients.Conclusion:The presence of mediastinal necrotic lymph node is the most accurate CT finding that can differentiate recurrent TB from post-TB sequelae. No other single chest CT scan finding had reliable diagnostic accuracy in comparison to microbiological tools in diagnosing recurrence among sputum negative or scarce previously treated PTB suspects.

Estimating SARS-CoV-2 Seroprevalence in Canadian Blood Donors, April 2020 to March 2021: Improving Accuracy with Multiple Assays.

ABSTRACTWe have previously used composite reference standards and latent class analysis (LCA) to evaluate the performance of laboratory assays in the presence of tarnished gold standards. Here, we apply these techniques to repeated, cross-sectional study of Canadian blood donors, whose sera underwent parallel testing with four separate SARS-CoV-2 antibody assays. We designed a repeated cross-sectional design with random cross-sectional sampling of all available retention samples (n = 1500/month) for a 12 -month period from April 2020 until March 2021. Each sample was evaluated for SARS-CoV-2 IgG antibodies using four assays an Abbott Architect assay targeting the nucleocapsid antigen (Abbott-NP, Abbott, Chicago IL) and three in-house IgG ELISAs recognizing distinct recombinant viral antigens: full-length spike glycoprotein (Spike), spike glycoprotein receptor binding domain (RBD) and nucleocapsid (NP). We used two analytic approaches to estimate SAR-CoV-2 seroprevalence: a composite reference standard and LCA. Using LCA to estimate true seropositivity status based on the results of the four antibody tests, we estimated that seroprevalence increased from 0.8% (95% CI: 0.5–1.4%) in April 2020 to 6.3% (95% CI: 5.1–7.6%) in March 2021. Our study provides further support for the use of LCA in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable.IMPORTANCE Here, we describe an approach to estimating seroprevalence in a low prevalence setting when multiple assays are available and yet no known gold standard exists. Because serological studies identify cases through both diagnostic testing and surveillance, and otherwise silent, unrecognized infections, serological data can be used to estimate the true infection fatality ratio of a disease. However, seroprevalence studies rely on assays with imperfect sensitivity and specificity. Seroreversion (loss of antibody response) also occurs over time, and with the advent of vaccination, distinction of antibody response resulting from vaccination as opposed to antibody response due to infection has posed an additional challenge. Our approach indicates that seroprevalence on Canadian blood donors by the end of March 2021was less than 10%. Our study supports the use of latent class analysis in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable.

Assessing the diagnostic accuracy of the Xpert MTB/RIF assay in detecting epididymal tuberculosis.

We analyzed 84 samples from patients with suspected epididymal TB or chronic epididymitis who received surgical treatment (epididymal resection or epididymis-testicular resection) at our hospital between July 1, 2016 and July 1, 2020. A composite reference standard (CRS) was used in this study. We determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of the Xpert MTB/RIF assay for epididymal TB and compared its diagnostic accuracy with that of the Mycobacterium tuberculosis (MTB) culture test in detecting epididymal TB. Three patients were excluded because of incomplete data. Comparing with the composite reference standard, the sensitivity, specificity, PPV, NPV, and AUC of Xpert MTB/RIF assay for epididymal TB were 80.95% (69.09-89.75%); 94.44% (72.71-99.86%); 98.08% (89.74-99.95%); and 58.62% (38.94-76.48%), respectively, with an AUC of 0.88 (95% confidence interval: 0.79-0.94). The diagnostic sensitivity of the Xpert MTB/RIF assay was significantly higher than that of the MTB culture test for epididymal TB. We recommend the Xpert MTB/RIF assay as a diagnostic method for the detection of epididymal TB.

Combination of Xpert® MTB/RIF and DetermineTM TB-LAM Ag improves the diagnosis of extrapulmonary tuberculosis at Jimma University Medical Center, Oromia, Ethiopia.

Ethiopia is one of the high burden countries for extrapulmonary tuberculosis (EPTB); however, the prompt diagnosis of EPTB remains challenging. This study is aimed to evaluate the diagnostic performance of Xpert MTB/RIF and DetermineTM TB-LAM Ag (TB-LAM) for the prompt diagnosis of EPTB in Ethiopia. A total of 147 presumptive EPTB patients, including 23 HIV- positive participants were enrolled. Extra-pulmonary samples were collected from all presumptive EPTB cases and tested for Mycobacterium tuberculosis complex (MTBC) using fluorescent microscopy, Xpert MTB/RIF, and culture. Additionally, urine samples were also collected from 126 participants and were tested by DetermineTM TB-LAM Ag (Alere Inc, Waltham, USA). The Sensitivity and specificity of Xpert and TB- LAM tests were calculated by comparing with a composite reference standard (CRS), which comprises smear microscopy, culture and response to empirical anti-TB treatment. Of 147 patients, 23 (15.6%) were confirmed EPTB cases (culture-positive), 14 (9.5%) were probable EPTB (clinically, radiologically or cytologically positive and received anti-TB treatment with good response), and 110 (74.8%) were classified as "non- TB" cases. Compared to the composite reference standard (CRS), the overall sensitivity and specificity of Xpert MTB/RIF were 43.2% and 100%, respectively with the highest sensitivity for Lymph node aspirate (85.7%) and lower sensitivity for pleural fluid (14.3%) and 100% specificity for all specimen types. The sensitivity and specificity of TB-LAM were 33.3% and 94.4% respectively with the highest sensitivity for HIV co-infected participants (83.3%). The sensitivity of the combination of Xpert MTB/RIF and TB-LAM tests regardless of HIV status was 61.1% whereas the sensitivity was improved to 83.3% for HIV-positive cases. TB-LAM alone has low sensitivity for EPTB diagnosis; however, the combination of TB-LAM and Xpert MTB/RIF improves the diagnosis of EPTB particularly for countries with high EPTB and HIV cases.

Clinical impact of rapid viral respiratory panel testing on pediatric critical care of patients with acute lower respiratory infection

BackgroundWe aimed to determine the impact of utilizing a rapid panel test of respiratory viral and atypical bacteria (FilmArray® Respiratory Panel, FA RP) on etiological diagnosis of acute lower respiratory infection (ALRI) and antimicrobial stewardship in critical care pediatric patients. MethodsProspective cohort study of patients aged<18 years with clinical diagnosis of ALRI that were admitted to the Pediatric Intensive Care Unit (PICU) of Hospital Sant Joan de Deu (Barcelona, Spain) during December 2015–February 2017. Patients were diagnosed by FA RP and by a bundle of routine microbiological assays. ResultsALRI viral and bacterial etiology was confirmed by a composite reference standard of routine microbiological assays in 72 (55.4%) and 15 (11.5%) respiratory samples, respectively, that were collected from 130 children (median age, 3.5 months, IQR 1.1–14.8 months; 54.6% male). Comparatively, FA RP use increased etiological confirmation of ALRI in up to 123 (94.6%) samples (p<0.001) but only determined a bacterial origin in 2 (1.5%). Availability of diagnostic results before patient discharge from the PICU rose from 65.4 to 38.5% (p<0.001). Use of the new panel test directly influenced antimicrobial stewardship in 11 (8.4%) episodes, leading to discontinuation of antiviral drugs (n=5), administration of targeted antibiotics (n=3), antiviral therapy start (n=2) and both targeted antibiotic administration and discontinuation of antiviral drugs (n=1). ConclusionFA RP contributed to improve etiological diagnosis of ALRI in a timely manner while enhancing a more rational use of antimicrobial drugs in critical care pediatric patients.

Utility of Lung Perfusion SPECT/CT in Detection of Pulmonary Thromboembolic Disease: Outcome Analysis.

PurposeTo evaluate the clinical outcome of Q-SPECT/CT in pulmonary thromboembolic disease.MethodsFrom Jan 2020 to Jan 2021, 30 consecutive patients (M:F = 8:22; median age = 52 year (21–89)) suspected of having acute pulmonary embolism (PE) or chronic thromboembolic pulmonary hypertension (CTEPH) were referred for non-contrasted Q-SPECT/CT. All patients were COVID-19 PCR negative. MSKCC Q-SPECT/CT and/or PISAPED criteria were used to determine the presence of thromboembolic disease in Q-SPECT/CT. Final diagnosis was made based on composite reference standards that included at least 2-month clinical cardiorespiratory assessment and follow-up imaging.ResultsQ-SPECT/CT was positive in 19 patients: indeterminate in 1 and 10 were negative. Three false positive cases were observed during follow-up. Of the remaining 16 true positives, all patients’ cardiorespiratory symptom were improved or stabilised after treatment with anticoagulants. The overall sensitivity, specificity, PPV, NPV and accuracy of Q-SPECT/CT were 100% (95% CI, 79.41–100%), 78.57% (95% CI, 49.20–95.34%), 84.21% (95% CI, 66.41–93.57%), 100% and 90.00% (95% CI, 73.47–97.89%) respectively.ConclusionsIn the current COVID-19 pandemic, Q-SPECT/CT can be an alternative modality to detect pulmonary thromboembolic disease. Normal Q-SPECT/CT excludes pulmonary thromboembolic disease with high degree of certainty. However, false positive has been observed.

Diagnostic accuracy of Xpert ultra for childhood tuberculosis: A preliminary systematic review and meta-analysis.

Diagnosis of childhood tuberculosis (TB) is challenging. Xpert MTB/RIF and the new version Xpert MTB/RIF Ultra (Ultra) are molecular tests currently used to rapidly identify the infection. We reviewed the literature for the accuracy of Ultra assay in the diagnosis of tuberculosis and rifampicin resistance in children. We conducted a full search in PubMed, Web of Science (WOS), Embase, and Scopus, up to April 2021. A bivariate random‐effects model was used to determine the pooled sensitivity and specificity of Ultra, with a 95% confidence interval (CI), compared with culturing and the composite reference standard (CRS). In the ten included studies (2,427 participants), the pooled Ultra sensitivity and specificity, in diagnosing pulmonary tuberculosis (PTB), were 78% (95% CI, 73–82) and 92% (95% CI, 91–94), respectively, against culture. Since a high heterogeneity was found between studies, we created subgroups based on different samples and ages. Ultra‐pooled sensitivity was consistently lower against CRS (95% CI, 35%, 32–38). Compared to Xpert MTB/RIF, Ultra sensitivity tended toward higher values (Ultra: 73%, 67%–78% vs. Xpert MTB/RIF: 66%, 60%–72%), but specificity was lower (Ultra: 95%, 94%–96% vs. Xpert MTB/RIF: 99%, 98%–99%). Ultra has improved the definitive diagnosis of PTB, particularly in subjects with paucibacillary TB, including children. The lower specificity could be due to the fact that culture is an imperfect reference standard. Further studies are needed to evaluate the accuracy of Ultra in the diagnosis of childhood TB.

Study of diagnostic utility of Xpert MTB/Rif test on pleural fluid in the evaluation of patients presenting with Pleural Tuberculosis in Nepal.

Existing tests for the diagnosis of pleural tuberculosis (TB) have major limitations in terms of accuracy, time to diagnosis and drug resistance testing. A test which can diagnose pleural TB and detect resistance, like Xpert MTB/Rif, would be optimal for rapid diagnosis and treatment. A prospective observational study was done in a tertiary care hospital in Eastern Nepal. Fifty-one patients with clinic-radiologic suspicion of pleural TB were included. The results of pleural fluid Xpert MTB/Rif were compared with two Composite Reference Standards. Composite Reference Standard-1 consisted of positive pleural fluid smear, positive culture, positive histology of pleural biopsy, and positive sputum results. Composite Reference Standard-2 included those with Composite Reference Standard-1 and those with high ADA values (>40 U/l) with response to anti-tubercular treatment at 8 weeks of follow-up. Thirty-six patients were diagnosed as Pleural TB. Nine fulfilled Composite Reference Standard-1. Pleural fluid Xpert MTB/Rif was positive in five cases with Composite Reference Standard-1 and nine cases with Composite Reference Standard-2. The sensitivity, specificity, positive predictive value and negative predictive value with reference to Composite Reference Standard-1 were 55.56%, 88.10%, 50%, and 90.24%, respectively. Using Composite Reference Standard-2 as reference, sensitivity, specificity, positive predictive value and negative predictive value were 25%, 93.33%, 90%, and 34.15%, respectively. Two cases were diagnosed Xpert Rif resistant on pleural fluid. Due to low sensitivity, the Xpert MTB/Rif test cannot be recommended as initial test of diagnosis in a high prevalence setting. At the same time its clinical utility lies in testing of patients suspected to have drug-resistant pleural tuberculosis.

Interest of routine MR spectroscopic techniques for differential diagnosis between radionecrosis and progression of brain tumor lesions

ObjectivesThe main objective of the study is to assess the feasibility and reproducibility of routine MRS to assist in the differential diagnosis between post-radiation necrosis and tumor progression. The secondary objective is to evaluate the accuracy of the method. MethodAn additional sequence of MRS was added to the standard protocol routinely used for patient follow-up. To assess discomfort a control group was formed. The time required to perform MRS and analysis of results, and data about artefacts and technical limitations were collected. MRS results analyzed independently by two neuroradiologists were compared. The diagnostic accuracy of MRS was calculated using a composite reference standard. ResultsThe experimental group included 38 patients, the control group 41. The discomfort felt during the examination, is not significantly different between the groups. The average quality of SRM is rated as low. The frequency of cerebral radionecrosis is 13 % based on the reference standard used, 54 % and 46 % based on MRS results for the two observers. The additional time is 19,5 min. There is strong inter-observer agreement. The sensitivity and specificity of MRS are respectively for the diagnosis of radionecrosis of 60 % and 45 % (PPV = 16 %NPV = 87 %), for the diagnosis of tumor tissue of 25 % and 94 % (PPV = 80 %NPV = 57%). ConclusionMRS is probably not applicable in routine clinical practice; however, in view of our results and the literature, in selected cases, it could be a support in the diagnosis of radionecrosis or brain tumor progression. Radionecrosis is probably underestimated.

Increased Detection of Mycobacterium tuberculosis Disease Using a Tissue-Based Laboratory-Developed Polymerase Chain Reaction Assay Compared to Standard Diagnostics.

Standard diagnostics for Mycobacterium tuberculosis (MTB) including acid-fast bacilli (AFB) smear and culture, and Xpert™ MTB/RIF real-time Polymerase Chain Reaction (RT-PCR; Xpert) have variable sensitivity and/or long turnaround times. We describe the clinical performance of a laboratory-developed tissue-based MTB PCR compared with AFB culture and Xpert using a composite reference standard (CRS). Over an 8-year period, MTB PCR was performed on pulmonary, pleural, or lymph node specimens for 36 patients. Of these, 11 met criteria for confirmed/probable MTB using CRS. MTB PCR was positive in 100% (11/11), AFB cultures were positive in 73% (8/11), and Xpert in 0% (0/4). MTB PCR was negative in 25 cases of "No MTB" (100% specific). The MTB PCR assay resulted faster than positive AFB culture (mean time 4.3 versus 21.2 days). Tissue-based MTB PCR was associated with increased and rapid detection of MTB, improving clinical sensitivity in strongly suspected MTB cases.

An outcome-based composite approach for the diagnosis of intestinal tuberculosis: a pilot study from a tertiary care centre in South India.

The diagnosis of intestinal TB (ITB) is challenging because of its overlapping features with Crohn's disease. This outcome-based study evaluated the combination of colonoscopy, histopathology, Xpert MTB/RIF and TB culture for best sensitivity and specificity. This was a four-year retrospective, observational study of 426 clinically suspected patients who underwent colonoscopy with biopsies for histopathology, Xpert MTB/RIF and TB culture. ITB was diagnosed using the composite reference standard (CRS), which comprised either histological features or culture or Xpert MTB/RIF positivity, and positive response to anti-tuberculous treatment on follow up. 35 (8.2%) patients were diagnosed with ITB. Histopathology had the highest sensitivity (91.4%) and negative predictive value (99.2%), MTB/RIF had the highest specificity (100%) and positive predictive value (100%). A combinatorial approach with Xpert MTB/RIF and histopathology had optimal diagnostic value (97%), approaching 100% sensitivity with culture. 40% of cases were diagnosed within 12 hours with Xpert MTB/RIF and 97% cases within three days. This combinatorial diagnostic model provides rapid and reliable diagnosis of ITB which may be useful in endemic areas.

Diagnostic accuracy of adenosine deaminase for pleural tuberculosis in a low prevalence setting: A machine learning approach within a 7-year prospective multi-center study.

To analyze the performance of adenosine deaminase in pleural fluid combined with other parameters routinely measured in clinical practice and assisted by machine learning algorithms for the diagnosis of pleural tuberculosis in a low prevalence setting, and secondly, to identify effusions that are non-tuberculous and most likely malignant. We prospectively analyzed 230 consecutive patients diagnosed with lymphocytic exudative pleural effusion from March 2013 to June 2020. Diagnosis according to the composite reference standard was achieved in all cases. Pre-test probability of pleural tuberculosis was 3.8% throughout the study period. Parameters included were: levels of adenosine deaminase, pH, glucose, proteins, and lactate dehydrogenase, red and white cell counts and lymphocyte percentage in pleural fluid, as well as age. We tested six different machine learning-based classifiers to categorize the patients. Two different classifications were performed: a) tuberculous/non-tuberculous and b) tuberculous/malignant/other. Out of a total of 230 patients with pleural effusion included in the study, 124 were diagnosed with malignant effusion and 44 with pleural tuberculosis, while 62 were given other diagnoses. In the tuberculous/non-tuberculous classification, and taking into account the validation predictions, the support vector machine yielded the best result: an AUC of 0.98, accuracy of 97%, sensitivity of 91%, and specificity of 98%, whilst in the tuberculous/malignant/other classification, this type of classifier yielded an overall accuracy of 80%. With this three-class classifier, the same sensitivity and specificity was achieved in the tuberculous/other classification, but it also allowed the correct classification of 90% of malignant cases. The level of adenosine deaminase in pleural fluid together with cell count, other routine biochemical parameters and age, combined with a machine-learning approach, is suitable for the diagnosis of pleural tuberculosis in a low prevalence scenario. Secondly, non-tuberculous effusions that are suspected to be malignant may also be identified with adequate accuracy.

Incremental yield of Xpert® MTB/RIF Ultra over Xpert® MTB/RIF in the diagnosis of extrapulmonary TB.

BACKGROUND: Extrapulmonary TB (EPTB) comprises approximately 15-20% of TB cases worldwide, and its diagnosis is difficult. The sensitivity of Xpert® MTB/RIF (Xpert) in the diagnosis of EPTB is low on account of its paucibacillary nature. Xpert® MTB/RIF Ultra (Ultra) was developed to improve sensitivity.OBJECTIVE: To compare the sensitivity of Ultra test with that of Xpert against MGIT™ (Mycobacteria Growth Indicator Tube) culture and a composite reference standard (CRS).METHODS: We recruited consecutive treatment-naïve patients with suspected EPTB. Demographic information, clinical and relevant laboratory data were collected.RESULTS: From January 2019 to November 2019, 210 patients provided 250 samples. Against MGIT culture, the sensitivity of Ultra was significantly higher than Xpert (72% vs. 51.1%; P = 0.04), the specificity was lower (87.8% vs. 95.1%). Against the CRS also, the sensitivity of Ultra was significantly higher than Xpert (45.4% vs. 25.2%; P = 0.002); however, the specificities were similar (98.2% vs. 99.1%). The trend towards higher sensitivity of Ultra compared to Xpert was seen in most of the individual samples. The sensitivities against MGIT and CRS were as follows: lymph node (68.1% vs. 31.8%; P = 0.01) and (59.5% vs. 23.8%; P = 0.001), pleural biopsy (80.0% on both; P = NS) and (53.8% vs. 46.2%; P = NS) and pleural fluid (66.7% vs. 50%; P = NS) and (22.5% vs. 9.6%; P = NS), respectively.CONCLUSIONS: Xpert Ultra showed a significantly higher sensitivity in diagnosing EPTB than Xpert.

Towards soil-transmitted helminths transmission interruption: The impact of diagnostic tools on infection prediction in a low intensity setting in Southern Mozambique.

World Health Organization goals against soil-transmitted helminthiases (STH) are pointing towards seeking their elimination as a public health problem: reducing to less than 2% the proportion of moderate and heavy infections. Some regions are reaching WHO goals, but transmission could rebound if strategies are discontinued without an epidemiological evaluation. For that, sensitive diagnostic methods to detect low intensity infections and localization of ongoing transmission are crucial. In this work, we estimated and compared the STH infection as obtained by different diagnostic methods in a low intensity setting. We conducted a cross-sectional study enrolling 792 participants from a district in Mozambique. Two stool samples from two consecutive days were collected from each participant. Samples were analysed by Telemann, Kato-Katz and qPCR for STH detection. We evaluated diagnostic sensitivity using a composite reference standard. By geostatistical methods, we estimated neighbourhood prevalence of at least one STH infection for each diagnostic method. We used environmental, demographical and socioeconomical indicators to account for any existing spatial heterogeneity in infection. qPCR was the most sensitive technique compared to composite reference standard: 92% (CI: 83%- 97%) for A. lumbricoides, 95% (CI: 88%- 98%) for T. trichiura and 95% (CI: 91%- 97%) for hookworm. qPCR also estimated the highest neighbourhood prevalences for at least one STH infection in a low intensity setting. While 10% of the neighbourhoods showed a prevalence above 20% when estimating with single Kato-Katz from one stool and Telemann from one stool, 86% of the neighbourhoods had a prevalence above 20% when estimating with qPCR. In low intensity settings, STH estimated prevalence of infection may be underestimated if based on Kato-Katz. qPCR diagnosis outperformed the microscopy methods. Thus, implementation of qPCR based predictive maps at STH control and elimination programmes would disclose hidden transmission and facilitate targeted interventions for transmission interruption.

Comparison of Multiplex Real-Time PCR and Droplet Digital PCR for Detection of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma

Plasma Epstein-Barr Virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma (NPC) and EBV-associated lymphoproliferative disorders. The ongoing NRG-HN001 randomized trial is testing one biomarker-directed treatment approach using real-time PCR (qPCR). Existing qPCR assays are limited by inter-laboratory imprecision secondary to external calibrators, and development of a standardized assay would facilitate reproducible quantitation. We hypothesized that droplet digital PCR (ddPCR) would have similar analytical sensitivity, precision, and accuracy relative to qPCR.We designed a multiplex ddPCR assay to detect two single-copy EBV targets (EBNA-1, EBER), a variable-repeat EBV target (BamHI-W), and a human internal control (RNaseP) in plasma across two reactions. This was compared against the NRG-HN001 qPCR assay, which detects EBNA-1, BamHI-W, and an internal control across two reactions. Quantitation for the ddPCR assay was based on Poisson statistics and automated threshold determination without external calibrators. Quantitation for the qPCR assay was based on two external calibrators traceable to the WHO EBV standard. Assays were compared by measuring sensitivity, specificity, 95% lower limit of detection (LLOD), lower limit of quantitation (LLOQ), accuracy (difference from nominal concentration), and precision (reproducibility). The WHO EBV standard was used as control material. Clinical specimens sent for routine plasma EBV testing were collected from patients with EBV-associated neoplasms. For clinical specimens, a composite reference standard was defined as positivity by at least two targets.The 95% LLOD for BamHI-W, EBNA-1, and EBER ddPCR were 1.49, 13.26, and 9.77 international units (IU) per well, respectively. Analytical sensitivity was not significantly different than BamHI-W qPCR (1.75 IU/well, P = 0.99) or EBNA-1 qPCR (7.58 IU/well, P = 0.60). The LLOQ for both assays was 100 IU/mL. Across the range of prognostic cutoffs for plasma EBV DNA copy number (1,500-40,000 IU/mL), within-laboratory precision was highest with BamHI-W qPCR (0.04-0.06) and ddPCR (0.04-0.08) and lower with EBNA-1 qPCR (0.04-0.14). Across this range, ddPCR had modestly improved accuracy (absolute mean difference from nominal, BamHI-W: 1104 IU/mL; EBER: 1206 IU/mL) relative to qPCR (BamHI-W: 1565 IU/mL; EBNA-1: 1874 IU/mL, P = 0.77 across platforms). Sensitivity (100% vs. 95%, P = 0.50) and specificity (100% vs. 100%) were similar between BamHI-W ddPCR and BamHI-W qPCR for clinical specimens. Sensitivity was lower for all single-copy targets (80-85%).A multiplex ddPCR amplifying single- and variable-copy EBV targets in plasma offers similar analytical performance as the existing NRG-HN001 qPCR assay without the need for external calibrators. Further research will inform whether inter-laboratory precision is superior with ddPCR, which may facilitate standardization of EBV DNA quantitation.

Detection of isoniazid, fluoroquinolone, ethionamide, amikacin, kanamycin, and capreomycin resistance by the Xpert MTB/XDR assay: a cross-sectional multicentre diagnostic accuracy study

The WHO End TB Strategy requires drug susceptibility testing and treatment of all people with tuberculosis, but second-line diagnostic testing with line-probe assays needs to be done in experienced laboratories with advanced infrastructure. Fewer than half of people with drug-resistant tuberculosis receive appropriate treatment. We assessed the diagnostic accuracy of the rapid Xpert MTB/XDR automated molecular assay (Cepheid, Sunnyvale, CA, USA) to overcome these limitations. We did a prospective study involving individuals presenting with pulmonary tuberculosis symptoms and at least one risk factor for drug resistance in four sites in India (New Delhi and Mumbai), Moldova, and South Africa between July 31, 2019, and March 21, 2020. The Xpert MTB/XDR assay was used as a reflex test to detect resistance to isoniazid, fluoroquinolones, ethionamide, amikacin, kanamycin, and capreomycin in adults with positive results for Mycobacterium tuberculosis complex on Xpert MTB/RIF or Ultra (Cepheid). Diagnostic performance was assessed against a composite reference standard of phenotypic drug-susceptibility testing and whole-genome sequencing. This study is registered with ClinicalTrials.gov, number NCT03728725. Of 710 participants, 611 (86%) had results from both Xpert MTB/XDR and the reference standard for any drug and were included in analysis. Sensitivity for Xpert MTB/XDR detection of resistance was 94% (460 of 488, 95% CI 92-96) for isoniazid, 94% (222 of 235, 90-96%) for fluoroquinolones, 54% (178 of 328, 50-61) for ethionamide, 73% (60 of 82, 62-81) for amikacin, 86% (181 of 210, 81-91) for kanamycin, and 61% (53 of 87, 49-70) for capreomycin. Specificity was 98-100% for all drugs. Performance was equivalent to that of line-probe assays. The non-determinate rate of Xpert MTB/XDR (ie, invalid M tuberculosis complex detection) was 2·96%. The Xpert MTB/XDR assay showed high diagnostic accuracy and met WHO's minimum target product profile criteria for a next-generation drug susceptibility test. The assay has the potential to diagnose drug-resistant tuberculosis rapidly and accurately and enable optimum treatment. German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade.

Performance of loop-mediated isothermal amplification assay for diagnosis of extrapulmonary tuberculosis and antituberculosis treatment initiation.

Extra-pulmonary TB(EPTB) accounts for 15-20% of total TB cases in India. Many cases remain undiagnosed due to poor sensitivity/long turn-around-time of conventional diagnostic tests. Molecular tests offer rapidity, improved sensitivity and exquisite specificity, but are expensive, require skilled manpower and enhanced laboratory infrastructure. Loop-mediated isothermal amplification (LAMP) assay is a unique, temperature-independent DNA amplification test facilitated by visual optic-readout. WHO has recommended use of LAMP for pulmonary TB diagnosis in 2016. For END-TB strategy to succeed, its necessary to capture all forms of TB. The aim of the study was to determine the sensitivity and specificity of LAMP assay against culture, Xpert MTB/RIF assay and Composite Reference Standard(CRS) for diagnosis of EPTB. In a cross-sectional study hundred consecutive EPTB specimens were processed for microscopy, culture, Xpert and LAMP assay. Standard formulae of sensitivity and specificity and McNemar chi square test of significance was applied. Hundred specimens included in the study were fluids(65), pus(19) and tissue(16). TB was detected in 38 specimens by any of the four methods. Positivity of microscopy-5%, culture-28%, Xpert-25% and LAMP-32%. Sensitivity and specificity of LAMP against culture was 85.71% and 88.89%; against Xpert was 88% and 86.67%; against CRS was 80% and 88.6% respectively. LAMP detected TB in 32 patients of which 28 were put on anti-TB treatment (ATT). Of the 62 patients with negative results in all the tests, 22 were put on ATT. LAMP has good sensitivity for EPTB diagnosis. Further studies are required to establish utility of LAMP as EPTB diagnostic tool.

Diagnostic performance of the Abbott RealTime MTB assay for tuberculosis diagnosis in people living with HIV

Strengthening tuberculosis diagnosis is an international priority and the advocacy for multi-disease testing devices raises the possibility of improving laboratory efficiency. However, the advantages of centralized platforms might not translate into real improvements under operational conditions. This study aimed to evaluate the field use of the Abbott RealTime MTB (RT-MTB) and Xpert MTB/RIF assays, in a large cohort of HIV-positive and TB presumptive cases in Southern Mozambique. Over a 6-month period, 255 HIV-positive TB presumptive cases were consecutively recruited in the high TB/HIV burden district of Manhiça. The diagnostic performance of both assays was evaluated against two different reference standards: a microbiological gold standard (MGS) and a composite reference standard (CRS). Results from the primary analysis (MGS) showed improved sensitivity (Se) and reduced specificity (Sp) for the Abbott RT-MTB assay compared to the Xpert MTB/RIF (RT-MTB Se: 0.92 (95% CI: 0.75;0.99) vs Xpert Se: 0.73 (95% CI: 0.52;0.88) p value = 0.06; RT-MTB Sp: 0.80 (0.72;0.86) vs Xpert Sp: 0.96 (0.92;0.99) p value < 0.001). The lower specificity may be due to cross-reactivity with non-tuberculous mycobacteria (NTMs), the detection of non-viable MTBC, or the identification of true TB cases missed by the gold standard.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence: Navigating the absence of a gold standard.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence studies bridge the gap left from case detection, to estimate the true burden of the COVID-19 pandemic. While multiple anti-SARS-CoV-2 immunoassays are available, no gold standard exists.MethodsThis serial cross-sectional study was conducted using plasma samples from 8999 healthy blood donors between April-September 2020. Each sample was tested by four assays: Abbott SARS-Cov-2 IgG assay, targeting nucleocapsid (Abbott-NP) and three in-house IgG ELISA assays (targeting spike glycoprotein, receptor binding domain, and nucleocapsid). Seroprevalence rates were compared using multiple composite reference standards and by a series of Bayesian Latent Class Models.ResultWe found 13 unique diagnostic phenotypes; only 32 samples (0.4%) were positive by all assays. None of the individual assays resulted in seroprevalence increasing monotonically over time. In contrast, by using the results from all assays, the Bayesian Latent Class Model with informative priors predicted seroprevalence increased from 0.7% (95% credible interval (95% CrI); 0.4, 1.0%) in April/May to 0.7% (95% CrI 0.5, 1.1%) in June/July to 0.9% (95% CrI 0.5, 1.3) in August/September. Assay characteristics varied over time. Overall Spike had the highest sensitivity (93.5% (95% CrI 88.7, 97.3%), while the sensitivity of the Abbott-NP assay waned from 77.3% (95% CrI 58.7, 92.5%) in April/May to 64.4% (95% CrI 45.6, 83.0) by August/September.DiscussionOur results confirmed very low seroprevalence after the first wave in Canada. Given the dynamic nature of this pandemic, Bayesian Latent Class Models can be used to correct for imperfect test characteristics and waning IgG antibody signals.